Product DescriptionABD-F has a benzofurazan moiety that produces a highly fluorescent compound through the reaction with a sulfhydryl group. The excitation and emission of the devivatized compound are 389 nm and 513 nm, respectively. The reaction rate of ABD-F is 30 times faster than that of SBD-F. ABD-F reactions with thiol compounds are completed within 5 minutes in aqueous conditions at 50ºC, pH 8. However, ABD-F does not react with alanine, proline, or cystine under these conditions. Its maximum fluorescence intensity can be observed at pH 2. In reversephase HPLC analysis, pre-labeled ABD-thiol compounds can be detected separately. The detection limits (S/N=3) are 0.6 pmol per injection for cysteine, 0.4 pmol per injection for glutathione, 1.9 pmol per injection for N-acetylcysteine, and 0.5 pmol per injection for cysteamine.
ABD Labeling Protocol1. To prepare sample solution, mix or dissolve a sample with 100 mM borate buffer, pH 8.0 containing 2 mM EDTA.2. Mix 500 μl of the sample solution and 500 μl of 1 mM ABD-F/100 mM borate buffer in a reaction vial.3. Heat the vial at 50ºC for 5 minutes and cool it on an ice bath.4. Add 300 μl of 100 mM HCl aqueous solution to the reaction mixture.5. Use this mixture for HPLC analysis to determine ABD-labeled compounds; excitation: 389 nm, emission: 513 nm.
1. T. Toyo’oka, et al., New Fluorogenic Reagent Having Halogenobenzofurazan Structure for Thiols: 4-(Aminosulfonyl)-7-fluoro-2, 1, 3-benzoxadiazole. Anal Chem. 1984;56:2461-2464.2. T. Toyo’oka, et al., Isolation and Characterization of Cysteine-Containing Regions of Proteins Using 4-(Aminosulfonyl)-7-fluoro-2, 1, 3-benzoxadiazole and High-Performance Liquid Chromatography. Anal Chem. 1985;57:1931-1937.3. T. Toyo’oka, et al., Amino Acid Composition Analysis of Minute Amounts of Cysteine-containing Proteins Using 4-(Aminosulfonyl)-7-fluoro-2, 1, 3-benzoxadiazole and 4-fluoro-7-nitro-2, 1, 3-benzoxadiazole in Combination with HPLC. Biomed Chromatogr. 1986;1:15-20.4. T. Toyo’oka, et al., Simultaneous Determination of Thiols and Disulfides by High-performance Liquid Chromatography with Fluorescence Detection. Anal Chim Acta. 1988;205:29-41.5. Y. Luo, et al., Antichymotrypsin interaction with chymotrypsin. Intermediates on the way to inhibited complex formation. J Biol Chem. 1999;274:17733-17741.
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